Smooth muscle cells form the contractile element found in involuntary contractile organs such as the gastrointestinal tract, the urogenital tract, the vasculature, and the small airways of the lung. Smooth muscle is capable of slow, sustained contractions that require less energy to maintain than other muscle types.
Smooth muscle cells are quite different from either skeletal or cardiac muscle cells, beginning with their smaller size. Additionally, no striations are seen in smooth muscle cells, and high-resolution microscopy reveals that smooth muscle cells lack the sarcomeric organization of other muscle cell types. The thick (myosin) and thin (actin) filaments are dispersed throughout the cytoplasm of smooth muscle cells, in contrast to the well-organized parallel bundles seen in skeletal and cardiac sarcomeres. This unique organization has the advantage of allowing smooth muscle cells to contract to less than ⅕ of their resting lengths (L0), compared with ½ L0 for cardiac cells.
The contractile apparatus of smooth muscle cells consists of thick filaments of smooth muscle myosin and thin filaments of actin coated with the smooth muscle isoform of tropomyosin. As in other muscle types, the level of intracellular free calcium regulates force production in smooth muscle. The molecular mechanism by which calcium controls force production is different in smooth muscle than in striated muscle.
Primary control of smooth muscle myosin activity is via calcium-dependent phosphorylation of myosin, changing inactive thick filaments into an active conformation that can interact with actin and produce force. This differs from skeletal and cardiac muscle, where regulation occurs at the level of actin thin filaments. In these tissues, tropomyosin and the regulatory troponin complex decorate actin filaments and control access to myosin binding sites, and thus force production, in a calcium-sensitive manner. In smooth muscle, there is evidence suggesting that some degree of thin filament regulation occurs in smooth muscle cells, but the regulatory proteins are different (caldesmon, calponin) and their role has been less well defined than for the troponin complex in skeletal and cardiac muscle.
Myosin is present in all muscle and non-muscle cells. Of the ten distinct classes of myosin in human cells, myosin-II is the form responsible for contraction of skeletal, cardiac, and smooth muscle. This form of myosin is significantly different in amino acid composition and in overall structure from myosins in the other nine distinct classes (Goodson and Spudich, 1993). Myosin-II consists of two globular head domains, called Subfragment-1 or S1, linked together by a long—helical coiled—coiled tail. Proteolysis of myosin generates either S1 or heavy meromyosin (HMM, a two-headed form with a truncated tail), depending on conditions. S1 contains the ATPase and actin-binding properties of the molecule. S1 has been shown to be sufficient to move actin filaments in vitro, and is therefore clearly the motor domain of the molecule.
Although myosin II isoforms from various tissues differ in a number of biological properties, they all share the same basic molecular structure as a dimer of two heavy chains (approximately 200 kDa) noncovlantly associated with two pairs of light chains (approximately 20 and 17 kDa). The two globular amino-terminal heads are tethered together by the carboxyl-terminal alpha-helical coiled-coil that forms a tail. The tails are involved in the assembly of myosin molecules into filaments, whereas the heads contain an actin-activated Mg2+-ATPase activity. Each myosin head can be divided by three protease-sensitive regions into peptides of approximately 25, 50, and 20 kDa. The more amino-terminal 25 kDa-50 kDa junction is close to the ATP binding region, whereas the actin-binding domain is near the 50 kDa-20 kDa junction.
The high-resolution crystal structure for skeletal S1 is known in both its putative pre-stroke and post-stroke states. The crystal structure of the recombinant chicken smooth muscle myosin motor domain has also been determined. S1 consists of a globular actin binding and nucleotide binding region known as the catalytic domain. This domain is attached at its carboxy-terminus to an alpha-helix that has two light chains of ˜20 kDa each wrapped around it. This light-chain binding domain of S1 is known as the lever arm. Upon transitioning from the pre-stroke to the post-stroke state of the S1, the lever arm swings through an angle of ˜90 degrees about a fulcrum point in the catalytic domain near the nucleotide-binding site. The “power stroke” is driven by the hydrolysis of ATP.
The other end of the myosin molecule is an alpha-helical coiled—coiled tail involved in self assembly of myosin molecules into bipolar thick filaments. These thick filaments interdigitate between thinner actin filaments, and the two filament systems slide past one another during contraction of the muscle. This filament sliding mechanism involves conformational changes in the myosin heads causing them to walk along the thin actin filaments at the expense of ATP hydrolysis.
Activation of smooth muscle myosin is via phosphorylation of the 20 kDa myosin light chain by myosin light chain kinase (MLCK). Calcium sensitivity is achieved by the absolute requirement for having a Ca2+-calmodulin complex bound to MLCK. An increase in intracellular calcium drives formation of a calcium-calmodulin complex, which is then competent to bind to MLCK and stimulate phosphorylation. When calcium levels fall, the intracellular concentration of the Ca2+-calmodulin complex drops and more Ca2+-calmodulin dissociates from MLCK, causing inactivation. Counterbalancing the action of MLCK is myosin phosphatase, which dephosphorylates the 20 kDa myosin light chain and inactivates myosin.
Myosin heavy chain (MyHC) has been studied at the molecular level in striated muscle, where each isoform is encoded by a different member of a multigene family. In contrast, smooth muscle myosin heavy chain isoforms are produced by alternate splicing of a single gene. Of particular interest is the splice varriant that occurs in the myosin head, at the 25/50 kda junction. B-isoforms contain an extra seven amino acid. insert at this junction; this insertion doubles the rate of ATP hydrolysis as well as the velocity of actin filaments in an in vitro motility assay over that of A-isoforms lacking the insert. Interestingly, although A- and B-isoforms are co-expressed in several smooth muscle types, there appears to be preferential expression of particular isoforms in different tissues. For example, the slower A-isoform predominates in vascular tissue, while the faster B-isoform is the major species in visceral tissues like bladder and intestine. This correlates with the much lower maximal speed of shortening seen for aortic versus intestinal smooth muscle.
In addition to this N-terminal diversity, there is also splice site variation in the C-terminal tail of the heavy chain, producing the SM-1 and SM-2 isoforms. The SM-2 isoform contains 9 unique amino acids, whereas the SM-1 isoform contains 43 unique amino acids at the C-terminus. The functional consequences of these C-terminal variations remain unclear, but expression studies indicate that there is differential expression in developing smooth muscle and cultured cells. SM-1 is expressed first in fetal rabbit development, followed by SM-2 expression in late fetal or early neonatal development. Studies of SM-1 and SM-2 isoform composition in different smooth muscle tissues have not established clear tissue expression patterns.
Although the art has provided some structural and functional data regarding certain non-primate homologs of SMMyHC genes, mRNAs, and encoded proteins, there is a need in the art for primate, and particularly human, SMMyHC isoform polynucleotide sequences, peptide sequences, isoform proteins, antibodies thereto, and the like, as well as methods employing the aforesaid. The present invention provides those embodiments and others useful to those skilled in the art.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application and are each incorporated herein by reference. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.